Natural and synthetic proteins offer an incomparable array of unique biological functions that may be exploited for human therapeutic applications. Their clinical utility, however, is often limited by biochemical instability, poor pharmacologic properties, and potential to induce adverse immunogenicity. Incorporation of biomolecules, such as proteins, in long-circulating vehicles with attached polyethylene glycol (PEG) polymer chains (i.e., PEGylated vehicles), such as nanoparticles, may mitigate such issues. However, the stable encapsulation of large quantities of functional proteins in PEGylated vehicles has proven to be challenging. Conventional encapsulation techniques, which were originally developed for small-molecule drug delivery, require the input of high energies and/or the use of organic solvents for particle formation, and are therefore unsuitable for use with biologically complex and more labile macromolecules.
In particular, examples of conventional encapsulation techniques may include thin-film rehydration, direct-hydration, and electro-formation, which may be used to encapsulate small molecules and proteins with unique biological function into polymersomes generated from poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD). For example, methylene blue (mBlue; Mw=319.85 g/mol) may be used as a model small molecule, and myoglobin (Mb; Mw=17,600 Da) may be used a model protein with unique biological function (i.e., oxygen storage). The efficiencies of encapsulating methylene blue and myoglobin into PEO-b-PBD using the thin-film rehydration and direct hydration techniques have been compared. In particular, quantification of the maximum encapsulation of fully functional myoglobin was based on a number of characteristics, using these established techniques. For example, the concentration and the reduction-oxidation reaction (“redox”) state of iron in the heme group of myoglobin were respectively measured using inductively coupled plasma optical emission spectroscopy (ICP-OES) and UV-Vis absorption spectroscopy (also referred to as spectrophotometry). The morphologies and stabilities of polymersome-encapsulated myoglobin (PEM) were respectively verified by cryogenic transmission electron microscopy (cryo-TEM) and by dynamic light scattering (DLS). Equilibrium oxygen binding and release at various partial pressures of oxygen were measured using a Hemeox analyzer. While the thin-film rehydration and direct hydration techniques allowed for successful methylene blue encapsulation, encapsulation of myoglobin was uniformly poor. Therefore, improved methods for generating PEM will be beneficial for human therapeutic applications.